Characterization of resistance and virulence factors in livestock-associated methicillin-resistant Staphylococcus aureus

The study investigated the economic concerns associated with livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in livestock (cow), examining its connection to severe infections, antimicrobial resistance (AMR), and virulence factors. The research, conducted in Edo State, Nigeria, analyzed 400 samples (200 rectal and 200 nasal swabs) collected between March 2018 and February 2019. MRSA prevalence was identified using conventional culture-based methods and polymerase chain reaction (PCR) techniques, revealing 63.5% (n = 254) for Staphylococcus aureus and 55% (n = 220) for MRSA. Of the 76 mecA-positive MRSA isolates, 64.5% (n = 49) exhibited multidrug resistance (MDR) while the remaining were sensitive to specific antimicrobials. Key virulence genes, such as PVL (81.6%; n = 62) and tsst-1 (44.7%; n = 34), were prevalent, along with AMR genes like mecC, tetM, ermA, ermC, vanA, and vanC. Staphylococcal chromosomal cassette mec (SCCmec) typing identified different types, notably II, IVa, and IVb. Biofilm formation, a crucial virulence factor varied in strength, is associated with icaA and icaB genes (p < 0.01). The findings highlighted substantial AMR and biofilm-forming capacity within LA-MRSA isolates, emphasizing the importance of ongoing surveillance for informed treatment strategies, AMR policies, and control measures against MDR staphylococcal infections.

Staphylococcus aureus (S. aureus) is a key pathogen responsible for severe infections, including osteomyelitis, pneumonia, and endocarditis, leading to conditions like sepsis and bacteremia 1 .Methicillin-resistant S. aureus (MRSA) exhibits reduced susceptibility to beta-lactam antibiotics, attributed to the replacement of antibioticsensitive penicillin-binding proteins (PBPs) with the acquired PBP2a 2 .This resistance mechanism is governed by staphylococcal chromosomal cassettes (SCCmec), housing mecA or mecC genes 3 .SCCmec subtypes I-V contribute significantly to antimicrobial resistance (AMR), harbouring genetic determinants for resistance to tetracyclines, beta-lactams, aminoglycosides, and macrolides, resulting in the rise of multidrug-resistant (MDR) MRSA 4 .MRSA, causing around 150,000 annual cases in Europe and 7,000 deaths, includes livestock-associated strains (LA-MRSA) found in cattle, pigs, and poultry, posing a zoonotic threat 5 .This transmission risk is prominent among individuals with substantial exposure, such as veterinarians and farmers 6 .Facilitation of the transfer of resistance genes occurs through horizontal gene transfer by the presence of AMR genes within mobile genetic elements (MGEs) like SCCmec, emphasizing the need to understand these molecular characteristics for effective control measures.
In the realm of LA-MRSA, the ST398 sequence type is frequently associated with human infections, highlighting a potential zoonotic transmission risk 7 .LA-MRSA demonstrates the ability to reacquire MRSA genes linked to human invasiveness, showcasing its adaptability across host environments 8 .Virulence factors like toxic shock syndrome toxin (tsst) and Panton-Valentine leukocidin (PVL) significantly impact MRSA pathogenicity.The tetracycline resistance gene tetM is notably prevalent in MDR MRSA strains, adding complexity to antibiotic resistance patterns 9 .Recognizing MRSA as an important pathogen, the World Health Organization (WHO) acknowledges its propensity to acquire AMR determinants, often associated with a diverse array of virulence factors.Crucially, MRSA strains inherently resist all β-lactam therapeutic agents, including carbapenems and cephalosporins 10 .The predominant antimicrobial agents administered for S. aureus infections contribute to the rising occurrence of AMR-related cases, emphasizing the consequences of misuse and unrestricted use in livestock farming 4 .
The imprudent usage of antimicrobial agents has fueled selective pressure, driving the surge in AMR, particularly in S. aureus, giving rise to MDR strains.Colonized mammals, including those in Nigeria, serve as reservoirs for both methicillin-susceptible S. aureus and MRSA 11 .MRSA hasnt been identified across diverse sources, including sheep, humans, slaughtered animals, raw milk, poultry, and more 2,[12][13][14][15] .Notably, a substantial gap in knowledge exists regarding the molecular characteristics of MRSA in Nigerian cows.Research indicates that animal-derived MRSA strains exhibit heightened resistance to various antibiotics compared to human-derived strains 3 , emphasizing the need for targeted understanding and intervention.Widespread usage of antimicrobials as preventive measures and growth promoters in the livestock industry has surged, with 131,109 tons of antibiotics used in 2013, projected to exceed 200,000 tons annually by 2030 16 .This extensive use raises concerns about antibiotic resistance, potentially causing 10 million annual fatalities by 2050 17 .Inefficient antibiotic absorption in livestock leads to the excretion of residues in biological waste, accumulating over time.This reckless antibiotic use contributes to a heightened prevalence of antibiotic-resistant bacteria in livestock settings, posing a significant risk to other ecosystems, including the human food chain 18 .
The surge in AMR bacteria led the European Union (EU) to ban antibiotic use as growth promoters in 2006.Nigeria's Ministry of Agriculture advises against antibiotics for enhanced egg production and animal growth, urging strict adherence to veterinarian prescriptions 19 .Despite recommendations, routine antibiotic use persists in veterinary practices for prophylaxis and infection treatment.In 2015, the EU reported 8361 tons of veterinary antimicrobial use; tetracycline and penicillin are the most commonly prescribed antibiotics for animals in food production 20 .This aligns with World Antimicrobial Awareness Week, which aims to raise awareness among stakeholders about practices to curb drug-resistant microorganisms.In Nigeria, lax enforcement allows livestock farmers unrestricted antibiotic access, contributing to indiscriminate purchases and fostering self-medication issues in human medicine 19 .S. aureus possesses the crucial ability to form biofilms, boosting resistance to microbial control in food-producing animals 2 .Biofilm activities involving Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) and regulated by the icaABCD operon contribute to the prevalence of S. aureus in animal-derived products 3 .While research on LA-MRSA and occupational risks is extensive 9 , Nigeria needs more comprehensive insights into its molecular traits.This study in Edo, Nigeria, delves into LA-MRSA's role in MRSA epidemiology in cows, characterizing strains through antimicrobial testing, genetic element examination, and virulence profiling.

Prevalence of S. aureus and MRSA from food-producing animals (cows)
From the 400 samples (200 nasal and rectal specimens) obtained from the same animal source, 254(63.5%)were positive for S. aureus, while 220(55%) were positive for MRSA based on cultural and biochemical characteristics and molecular detection of specific marker genes.From the 200 rectal specimens, 143(71.5%)were positive for S. aureus, while 122(61%) were positive for MRSA.From the 200 nasal specimens, 111(55.5%)were positive for S. aureus, while 98(49%) were positive for MRSA (Table 1).A total of 76 MRSA isolates (38 nasal and 38 rectal) were selected and characterized further.All 76 isolates also harboured the mecA gene for MRSA confirmation.The methicillin-sensitive S. aureus was not characterized further.

Antimicrobial susceptibility profile of the MRSA isolates
All 38 (100%) nasal and rectal isolates exhibited resistance to penicillin, ceftaroline, and cefotaxime (

Discussion
AMR, inherent but exacerbated by excessive use, poses a global threat.Nigeria's Ministry of Agriculture warns of its impact on food security and global health 19 .Our study's S. aureus rates align with those from Denmark, while higher rates are noted from studies in Norway and the USA and lower rates from studies in Spain, South Africa, and Tunisia 21,22 .Our MRSA prevalence mirrors those from Latvia, contrasting lower rates from those in Portugal, Indonesia, Mexico, Nigeria, and Korea 1,11 .Elevated S. aureus and MRSA prevalence in our pastoralfocused study suggest influences from unmonitored feeds, water exposure, varied environmental factors, treatment, breeding systems, sanitation, seasons, location, and animal species 21 .Intensifying surveillance of AMR in animal populations, particularly those integral to the food supply, is critical, especially in developing nations with limited epidemiological data.Our research identifies MDR MRSA, reinforcing our earlier findings 13,14 .These strains pose risks to both animal and human health through products from colonized animals, aligning with studies highlighting food-producing animals as significant AMR MRSA reservoirs.The potential circulation of these strains between animals and humans is substantial 6 .Penicillin resistance (100%) in our study mirrors findings in Nigeria, Bangladesh, and Ethiopia, while lower rates (72.6-98.5%)were reported in India, Ethiopia, and China 23,24 .www.nature.com/scientificreports/ In contrast to our findings, a study reported high sensitivity (96%) of S. aureus of cattle origin to cefotaxime and ceftaroline 25 .However, consistent with our research, another study revealed universal resistance of S. aureus isolates to cefotaxime 26 .This emphasizes the crucial role of veterinarians in judicious antibiotic use for foodproducing animals, advocating for prescription-based administration to treat infections 19 .Contrary to our study, Naas et al. 26 reported higher clindamycin resistance, and Kou et al. 24 found MRSA more sensitive to clindamycin.The inefficacy of penicillins, cephalosporins, and carbapenems against S. aureus has been noted globally, linked to their extensive use in veterinary medicine 4 .Resistance patterns vary regionally and are closely tied to levels of antimicrobial usage (AMU).LA-S.aureus strains, according to Omwenga et al. 4 , primarily resist tetracycline and ampicillin.Our study identified significant tetracycline resistance, although lower than Onyenwe et al. 25 and Gali et al. 23 , but higher than Kou et al. 24 .Fluoroquinolone resistance aligns with previous research 11,27 .MRSA isolates in our study moderately responded to kanamycin, gentamicin, and vancomycin, consistent with Onyenwe et al. 25 and Kou et al. 24 but not Naas et al. 26 .Vancomycin's comparatively higher effectiveness might stem from more judicious use in the study area 28 .Bernier-Lachance et al. 3 found sensitivity to various antibiotics, with only certain classes showing no resistance in our study.
In contrast to Silva et al. 11 , our MRSA isolates did not universally resist penicillin, ciprofloxacin, clindamycin, and erythromycin.Similar to Back et al. 1 , MRSA strains in our study resisted ampicillin but were sensitive to teicoplanin.Variable resistance rates are linked to geographic differences in AMU 4 .Our investigation revealed a MAR index ranging from 0.15 to 70, surpassing the 0.2 threshold, indicating inappropriate antibiotic use and posing risks to public health 29 .
The excessive use of antibiotics in livestock, observed in our study area, may be a key driver in the selection of AMR strains.The diverse resistance patterns identified in our research can be attributed to the prolonged and indiscriminate application of various antimicrobial classes in modern agriculture, as highlighted in previous  www.nature.com/scientificreports/studies 27,30 .Bernier-Lachance et al. 3 findings, where all isolates exhibited intermediate or full resistance to β-lactams, align with our results.While our study reported lower levels of MDR compared to other research 3,11 , Omwenga et al. 4 identified 94% of MDR S. aureus isolates as MRSA, inherently resistant to commonly prescribed antibiotics.Back et al. 1 proposed that the co-selection pressure linked to the MDR phenotype significantly contributed to the emergence and continuity of LA-MRSA.The surge in MDR MRSA is linked to extensive antibiotic use in veterinary and human healthcare 4 .Genetic factors carried by resistant isolates contribute to observed resistance patterns, highlighting the pivotal role of selective pressure from antibiotics 31,32 .The dissemination of AMR in S. aureus involves various MGEs 14 .Recognizing the genetic factors linked to resistance phenotypes is essential for comprehending the molecular mechanisms underlying the rise and dissemination of AMR.The genetic profiles of resistance phenotypes closely align with resistant isolates, suggesting that genes carried by LA-MRSA strains may underpin observed resistance phenotypes.
In this study, the identification of the mecA gene in all LA-MRSA isolates, as reported in the Silva et al. 11 investigation from Quails in Portugal as well Gaddafi et al. 33 from dairy cows in Nigeria, indicates its consistent presence and remains the definitive indicator for MRSA detection.mecA-positive staphylococcal strains, immune to all β-lactam antibiotics, highlight the significance of this genetic marker 34 .Using PCR for MRSA detection mitigates the risk of false-negative results common with phenotypic methods.Instances of mecA-positive S. aureus strains with phenotypic oxacillin susceptibility have been documented, emphasizing the need for a combined approach evaluating both phenotype and genotype to prevent false outcomes in MRSA identification 35 .Our study revealed that 81.6% of MRSA isolates were categorized as community-acquired MRSA (CA-MRSA) due to the presence of PVL genes.This rate surpasses previous findings 36 and falls below others 3 .PVL genes, virulence factors associated with CA-MRSA, enhance virulence and serve as genetic markers, differentiating them from hospital-associated MRSA 37 .Notably, PVL and human-related immune evasion cluster genes were absent in ST398 LA-MRSA strains in previous studies 7,11 .The acquisition of PVL and TSST-1 genes in LA-MRSA poses a considerable threat to public health.Bernier-Lachance et al. 3 reported negative results for tsst-1 alleles, contrasting with our findings.
Apart from the mecA gene, the MRSA isolates in our study contained genes associated with resistance to tetracyclines, lincosamides, and macrolides, consistent with findings in previous research 20 .The presence of these genes aligns with observed phenotypic AMR.Correlations were identified, such as tetracycline use with tetM detection, penicillins with mecA/mecC, vancomycin with vanA and vanC, and macrolides with ermA and ermC, as reported elsewhere 4,11 .The occurrence of MDR S. aureus and tetM detection has been linked to AMU 4 .Our study detected ermA in proportions comparable to studies in Kenya but lower than in Egypt, with erm genes facilitating adjustments to 23S rRNA 38 .The administration of tylosin might influence resistance to macrolides for treating prevalent diseases in the region.Similar to Silva et al. 11 , our study identified macrolide-lincosamide resistance genes, including ermC or a combination of ermB and ermC.Tetracycline resistance genes, including tetM, were found, which is consistent with Silva et al. 11 .Tetracycline and penicillin, frequently used in livestock farming, contribute to the prevalence of these genes 20 .The tetM genes are typically located in transposons or chromosomes 39 .Resistance determinants often cluster on MGEs, intensifying co-selection and resulting in coresistance to other antibiotics.Plasmids carrying tetracycline-resistant genes may also harbour supplementary genes, and the extensive utilization of antibiotics with broad-spectrum activity could contribute to the coselection of resistance genes.
A previous study 9 reported a higher prevalence of SCCmec gene cassette type V compared to type I among LA-MRSA strains, diverging from our findings.A study from Greece identified SCCmec type V elements in both human and LA-MRSA isolates 7 .The predominant SCCmec type V in CC398 LA-MRSA typically carries genes detoxifying heavy metals like cadmium and copper, commonly used in farm growth promotion 40 .Copperresistant genes contribute to bacterial survival 41 .ST398 isolates contained SCCmec type V, while ST6831 was untypeable 11 .ST398 and ST541 MRSA isolates, except for two non-typeable LA-MRSA, carried SCCmec type V 1 .Other MRSA isolates with non-CC398 genotypes had SCCmec IV or SCCmec II.Although MLST was not conducted, certain isolates in our study contained SCCmec types II, III, IVa, IVb, IVd, IVh, and V. Isolates with SCCmec V also proportionally carried tetK 42 .The co-localization of czrC and mecA genes in SCCmec V suggests zinc inclusion in livestock feed might promote czr in CC398 LA-MRSA 1 .
S. aureus's biofilm-forming capability, a crucial virulence factor, intensifies AMR, posing formidable challenges in infection management 2,43 .Studies highlight that biofilm-forming strains exhibit elevated MDR and methicillin resistance compared to non-biofilm strains 44 .Biofilms, with their adherence to diverse surfaces, enhance antibiotic resistance and survival in varied environments 29,45,46 .In contrast, Bernier-Lachance et al. 3 found that all isolates formed biofilms, with biofilm-forming isolates possessing icaACD genes, which are essential for biofilm formation.In our study, some isolates formed biofilms carrying only icaA and icaB genes.Positive MSCRAMMs gene probes in MRSA indicate their genetic capability for efficient adhesion, promoting biofilm formation, potentially enhancing persistence colonization, and facilitating zoonotic transmission 3 .Key limitations of the study include limited sampling diversity as it did not include humans and the environment, lack of investigation into transmission pathways involving potential LA-MRSA transmission between livestock and humans or other environmental reservoirs, limited exploration of antimicrobial resistance mechanisms with exploring the resistance evolution and transmission dynamics, and reliance on conventional methods such as PCR techniques, without exploring complementary approaches, such as whole-genome sequencing.

Conclusion
Detecting MDR MRSA in livestock raises health concerns for handlers and the community.Improving hygiene around food-producing animals is crucial to mitigate microbiological risks.Many isolates in our study showed heightened AMR and robust biofilm-forming abilities.Routine monitoring of biofilm development and AMR in S. aureus is essential for effective treatment and AMR control.Implementing immunizations, proper animal care, and biosecurity measures can reduce infections and antibiotic use in livestock.Governments and policymakers must lead in establishing sustainable policies to curb indiscriminate antibiotic use, promoting both human and animal health.

Study area and sample collection
Samples were obtained from free-range cows in Edo State.The farmers commonly administered tetracyclines and penicillin as the predominant antibiotics for livestock.Other antibiotics, such as fluoroquinolones, sulphonamides, cephalosporins and streptomycin, are also used.Single (oral, topical or parenteral) preparations of the antibiotics were mostly observed during field analysis, followed by > 1 preparation, while a combination of preparations of antibiotics was less commonly observed.The animal handlers usually source antibiotics from veterinary pharmacy shops, veterinary clinics, market displays, and drug hawkers.Non-antibiotic drugs such as multivitamins, anthelminthic drugs, traditional concoctions, salts, ashes, pepper, onion, potash and herbs were also observed to be used.Information gathered revealed that the antibiotics used were recommendations by veterinarians, from personal experience, on recommendations by drug sellers, on advice from fellow farmers, or based on advertisements.
The study adopts a longitudinal design, wherein animals serve as their controls.The sampling procedures adhered to the guidelines set by the European Food Safety Authority 47 .The sample size for this study was determined using the formula: where: Z 1-α/2 = Standard normal variant at 5% type I error (P < 0.05), P = Expected prevalence based on previous studies (ranging from 3.4% to 35.7%) 1, [22][23][24]28,29,36 , d = Absolute error or precision (set at 5%).
Based on this calculation, the expected maximum number of samples was 353.However, a total of 400 swab samples, including 200 rectal and 200 nasal samples, were systematically collected from free-range cows in Edo State, Nigeria, from March 2018 to February 2019.The rectal and nasal samples were taken from the same cows.The swab samples were acquired from the cows using sterile swab sticks moistened with normal saline, ensuring a well-organized approach to prevent any duplication of samples.Subsequently, the collected samples were promptly transported to the laboratory of the Applied Microbial Processes and Environmental Health Research Group (AMPEHRG) at the University of Benin, located in Benin City, Nigeria.The samples were kept on ice during transport to facilitate the subsequent processing.In addition to the sample collection, information regarding the treatment history and the types of antibiotics used was collected to provide a comprehensive description of the study population.

Ethics declaration
All methods described in this study were conducted following the guidelines and regulations outlined by relevant institutional and national committees for research involving animals and human subjects.The research protocols were reviewed and approved by the Research and Ethics Committee, State Ministry of Health, Edo State, Nigeria, with reference number Ha.737/5/T1/019 before the commencement of the study.Additionally, we confirm that all methods reported in this manuscript adhere to the guidelines provided by the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines (https:// arriv eguid elines.org) for reporting experiments involving animals.

Laboratory identification of S. aureus and MRSA
The swab sticks were introduced into tryptone soy broth (Merck, Darmstadt, Germany) and subsequently placed in an incubator at 37 °C for a duration of 18 to 24 h.Following incubation, the broth was subjected to a streaking technique on both Baird-Parker agar (Merck, Darmstadt, Germany) and MRSA selective agar plates (CHRO-MagarTM MRSA-ITK Diagnostics BV, Netherlands).These plates were then incubated at 37 °C for 24 h.Any colonies exhibiting circular, smooth, convex, moist, grey to jet-black characteristics on Baird-Parker agar, as well as rose to mauve colonies on MRSA selective agar plates, were considered as presumptive S. aureus and presumptive MRSA, respectively.To further confirm the identity of these colonies, one colony from each plate was isolated and purified by cultivation in nutrient agar (Lab M, Lancashire, United Kingdom).These colonies were then subjected to another 18-h incubation at 37 °C and subsequently stored at 4 °C on nutrient agar slants.The identification of the isolates was based on a series of cultural, morphological, and biochemical tests, including Gram staining, 3% potassium hydroxide testing, coagulase, catalase, and anaerobic utilization of mannitol and glucose, following the procedures outlined by Tallent et al. 48.Only the MRSA isolates underwent further characterization.A positive control in the form of S. aureus (ATCC 12600) was employed for reference purposes.

DNA extraction and molecular characterization of the MRSA isolates
DNA extraction was conducted following a previously established protocol 30,49 .To resuscitate all isolates, they were placed in 5 mL of tryptone soy broth after an initial 24-h incubation at 37 °C.Following this, cells were collected by centrifuging 2 mL of the incubated broth in sterile Eppendorf tubes at 5,000 rpm for 10 min.The cell deposit underwent a washing step with normal saline after discarding the supernatant, followed by re-centrifugation at 5000 rpm for 3 min.The cell pellet was subsequently re-suspended in a microcentrifuge tube containing a rapid lysis buffer.This lysis buffer included the following components: 100 mM NaCl, 10 mM Tris-HCl at pH Vol:.( 1234567890 www.nature.com/scientificreports/8.3, 1 mM EDTA at pH 9.0, and 1% Triton X-100.The mixture was boiled for 15 min, and subsequently, it was centrifuged at 10,000 rpm.The resulting supernatant was collected and stored at − 20 °C for future use as the template DNA.To identify S. aureus (based on the nuc gene), a polymerase chain reaction (PCR) was conducted using specific primers, as previously reported by Brakstad et al. 50, with S. aureus ATCC 12600 serving as the positive control.The specific primer used for identification is detailed in Supplementary Table 1 for the amplification of AMR genes, such as methicillin resistance (mecA) that was also used as a determinant to confirm the presumptive MRSA isolates molecularly, tetracycline (tetM), erythromycins (ermA, ermC), vancomycin (vanA, vanC), as well as virulence genes including PVL, tsst-1, intercellular adhesion proteins (icaA and icaB), procedures outlined in prior studies [51][52][53] were followed.SCCmec typing of MRSA isolates involved PCR amplification of SCCmec types 1 to V and subtype SCCmec (Iva to d) as described by Okuma et al. 54 , Ma et al. 55 , and Zhang et al. 56 .Specific primer sets listed in Supplementary Table 1 were used.The PCR products were electrophoresed on a 1% agarose gel at 110 V for 45 min and visualized after ethidium bromide staining using a transilluminator (Vilber Lourmat, EBOX VX5, France).

The biofilm formation profile of MRSA isolates
After the molecular confirmation of the MRSA isolates, pure MRSA colonies were introduced into 4.5 mL of tryptone soy broth (TSB) and then subjected to incubation at 37 °C for 18 h.Subsequently, these cultures underwent centrifugation for 2 min at 12,000 rpm.The resulting cell pellets were washed and re-suspended in phosphate-buffered solution (PBS) adjusted to pH 7.2, targeting 0.5 McFarland standards.The prepared suspension inoculants were introduced into the wells of sterile 96-well polystyrene microtiter plates containing 20 mL of cell suspensions and 180 mL of TSB to investigate the adherence of Staphylococci to a solid surface, following the procedure described previously 30 .The negative control wells contained only TSB broth, while S. aureus ATCC 12600 served as the positive control.Each of the assays was performed in triplicate to calculate the mean value.Based on previously established protocols 30 , biofilm formation was classified as non-producing/negative (ODi < ODc), weak/poor-producing (ODc < ODi < 0.1), moderate/intermediate-producing (0.1 < ODi < 0.12), or strong producer (ODi > 0.12).

Antimicrobial susceptibility profile of the MRSA isolates
Antimicrobial susceptibility testing was conducted through the Kirby-Bauer disk diffusion method.To do this, a suspension of the test isolates was prepared at a 0.5 McFarland standard.Aseptically, a suspension of the isolated test strains was streaked on Mueller-Hinton agar (obtained from Lab M, Lancashire, UK), and the corresponding antibiotic discs were aseptically positioned on the inoculated agar.The antibiotic discs used in this study were all sourced from Mast Diagnostics, United Kingdom.The disc employed includes penicillin G (PEN) (10 units), piperacillin (PTZ) (100 µg), gentamicin (GEN) (10 µg), kanamycin (KAN) (30 µg), tetracycline (TET) (30 µg), imipenem (IMP) (10 µg), meropenem (MEM) (10 µg), ertapenem (ETP) (10 µg), ceftaroline (CRO) (30 µg), cefotaxime (CTX) (30 µg), erythromycin (ERY) (15 µg), clindamycin (CLI) (30 µg), ciprofloxacin (CIP) (10 µg), quinupristin-dalfopristin (QDA) (15 µg), and linezolid (LIZ) (30 µg).The agar plates were allowed to air-dry at room temperature for approximately 10 min, followed by an incubation at 37 °C for 24 h.The diameter of the inhibition zone was measured using a transparent meter rule and interpreted according to recognized criteria, categorizing the strains as sensitive (S), intermediate resistant (I), or resistant (R) in accordance with the standards recommended by the Clinical Laboratory Standards Institute 57 .The minimum inhibitory concentration (MIC) protocol was carried out by preparing stock solutions of each of the antibiotics at various concentrations (vancomycin 1-32 µg/mL, oritavancin 0.12-0.50µg/mL, teicoplanin 4-64 µg/mL, daptomycin 0.5-4 µg/mL, and tedizolid 0.25-4 µg/mL), typically using serial dilutions.The standardized bacterial suspension was inoculated onto Mueller-Hinton broth (obtained from Lab M, Lancashire, UK).The antibiotic solutions were added to the bacterial cultures, ensuring that each well on the 96 well microtiter plate contained a different concentration of the antibiotic being tested.The broth cultures were incubated at 37 °C for 24 h.After incubation, bacterial growth was assessed in each well.The MIC is defined as the lowest concentration of antibiotic that completely inhibits visible bacterial growth.The MIC values obtained were compared with established interpretive guidelines and MIC breakpoints provided by the CLSI 57 .This helped to determine whether the bacteria were susceptible, intermediate, or resistant to the antibiotics being tested.. S. aureus ATCC 12600 was used as a positive control in this experiment.

Statistical analysis
All data in this research were subjected to analysis utilizing the statistical package SPSS version 21.0 and Microsoft Excel 2013.Descriptive statistics, which included the calculation of means and standard deviations, were employed for data summarization.A One-Way Analysis of Variance (ANOVA) was employed to analyze multiple variables, with the Duncan Multiple Range test used to identify significant differences between means.A probability value below 0.05 was regarded as indicative of statistical significance.

Figure 2 .
Figure 2. Distribution of SCCmec gene types in the staphylococcal isolates.

Figure 3 .
Figure 3. Distribution of biofilm formation in the staphylococcal isolates.